Stabilized liquid formulations containing receptors

ABSTRACT

Methods and reagents are disclosed for preparing a liquid solution of a receptor. The methods comprise combining in a liquid medium the receptor, a chelating agent and a C2-C6 polyol. An amount of the chelating agent and the C2-C6 polyol is sufficient to achieve a stable and active receptor in the liquid solution, which is maintained at a temperature of about 2° C. to about 40° C. The compositions may be employed in assays for the determination of analytes that include receptor-binding analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/823,116 filed May 14, 2013, which is incorporated herein byreference in its entirety.

BACKGROUND

The invention relates to compositions, methods and kits for thedetermination of analytes such as, for example, antibodies, in samples,such as patient samples, known or suspected to contain one or more ofsuch analytes. In some examples the invention relates more particularlyto maintaining stability of receptor reagents during storage as liquidformulations at temperatures above freezing. In some examples theinvention relates more particularly to enhancing the sensitivity ofreceptor reagents for use in assays for analytes.

The clinical diagnostic field has seen a broad expansion over the past35 years or more, both as to the variety of materials of interest thatmay be readily and accurately determined, as well as the methods for thedetermination. Convenient, reliable and non-hazardous means fordetecting the presence of low concentrations of materials in liquids isdesired. In clinical chemistry these materials may be present in bodyfluids in concentrations below 10⁻¹² molar. The difficulty of detectinglow concentrations of these materials is enhanced by the relativelysmall sample sizes that can be utilized.

The need to determine many analytes in blood and other biological fluidshas become increasingly apparent in many branches of medicine. Inendocrinology the knowledge of plasma concentration of a number ofdifferent hormones is often required to resolve a diagnostic problem ora panel of markers for a given diagnosis where the ratios could assistin determining disease progression. Receptors such as hormone receptorsare used commonly in a number of in vitro applications. In diagnosticassays, for example, receptors such as, for example, hormone receptorsare used for detection of patient antibodies that are indicative of oneor more disease states. In a particular example, thyrotropin (also knownas thyroid-stimulating hormone or TSH) receptors are used in diagnosticsassays for the detection of TSH receptor antibodies as an aid in thedifferential diagnosis of Graves' disease.

Due to their instability in liquid form, many receptors are either driedon a surface or lyophilized. These approaches are not desirable sincethey add manufacturing cost and are inconvenient for those who use thereagents. Furthermore, upon reconstitution for use, many such receptorsstill have a very short half-life.

There is, therefore, a continuing need to develop compositionscomprising receptors that exhibit good stability in a liquid form andgood sensitivity in an assay employing the receptors to measure levelsof antibodies and other analytes in patients.

SUMMARY

Some examples in accordance with the principles described herein aredirected to methods of preparing a liquid solution of a receptor. Themethods comprise combining in a liquid medium the receptor, a chelatingagent and a C2-C6 polyol. An amount of the chelating agent and the C2-C6polyol is sufficient to achieve a stable and sensitive receptor in theliquid solution, which is maintained at a temperature of about 2° C. toabout 40° C. during storage.

Some examples in accordance with the principles described herein aredirected to methods of stabilizing a liquid solution of a thyroidstimulating hormone receptor. The method comprises combining a liquidsolution comprising a thyroid stimulating hormone receptor with astabilizing amount of both (i) a chelating agent selected from the groupconsisting of triacetic acid chelating agents and tetraacetic acidchelating agents and (i) a C3-C5 polyol.

Some examples in accordance with the principles described herein aredirected to compositions comprising an aqueous medium, a receptorchimera, a chelating agent in an amount of about 0.1 mM to about 20 mM,and a C2-C6 polyol in an amount of about 5% to about 50% by weight.

Some examples in accordance with the principles described herein aredirected to methods of detecting a thyroid stimulating hormone receptorantibody in a sample. A combination is provided in an assay medium wherethe combination comprises a sample suspected of containing a thyroidstimulating hormone receptor antibody, a first thyroid stimulatinghormone receptor bound to a support and a second thyroid stimulatinghormone receptor bound to a label. Either or both of the first andsecond thyroid stimulating hormone receptors are from compositionsstored in accordance with the principles described herein. Thecombination is examined for the formation of a complex comprising thethyroid stimulating hormone receptor antibody. The presence of thecomplex is related to one or both of a presence and an amount of thethyroid stimulating hormone receptor antibody in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings provided herein are not to scale and are provided for thepurpose of facilitating the understanding of certain examples inaccordance with the principles described herein and are provided by wayof illustration and not limitation on the scope of the appended claims.

FIG. 1 is a graph depicting a plot of signal versus pH for a receptorchimera conjugated to alkaline phosphatase (the “conjugate”) upondissolution in citrate buffer in an example of a method in accordancewith the principles described herein and also, for purposes ofcomparison, upon dissolution of the conjugate in MES and PIPES buffers,respectively.

FIG. 2 is a graph depicting a plot of percent signal retained versus pHfor the conjugate solutions of FIG. 1 after storage at 37° C. for oneday.

DETAILED DESCRIPTION

General Discussion

The disclosure in accordance with the principles described hereinprovides methods of preparing liquid formulations of receptors that maybe stored for extended periods of time while maintaining the stabilityof the receptor. In addition, the present inventors have discovered thatthe sensitivity of receptors prepared in accordance with the principlesdescribed herein is enhanced over that of receptors subjected to dryingor lyophilization and subsequent reconstitution. As mentioned above,some examples in accordance with the principles described herein aredirected to methods of preparing a liquid solution of a receptor,storage of such liquid solutions, and use of such liquid solutions inassays for detection of analytes of interest.

DETAILED DISCUSSION

The term “receptor” as used herein refers to compounds that bind with asubstance (sometimes referred to as a ligand) for which they arestructurally specific. The receptors have an area on the surface or in acavity, which specifically binds to and is thereby defined ascomplementary with a particular spatial and polar organization ofanother molecule sometimes referred to as a ligand. Ligands may be smallmolecules including, but not limited to, drugs, small peptides, orsteroids, for example, or large molecules such as large peptides,proteins including antibodies, hormones, DNA, RNA, carbohydrates, forexample, and including portions or regions thereof. By way ofillustration and not limitation, receptors include hormone receptors,drug receptors, and antigen receptors, for example. The receptor may bean intact or complete receptor or a fragment or segment thereof.Fragments or segments may be prepared by subjecting an intact receptorto fragmentation techniques. On the other hand, fragments or segments ofreceptors, as well as intact or complete receptors, may be preparedsynthetically. Thus, the receptor may be naturally occurring andisolated from a source or the receptor may be synthetic and prepared bysynthetic techniques that include, but are not limited to, recombinanttechniques and chimeric techniques, for example.

In some examples the receptors are those that are specific forantibodies such as, for example, autoantibodies that are associated withcertain disease states, which include, but are not limited to,dermatomyositis, diabetes mellitus, epilepsy, Kawasaki's disease,glomerulonephritis, Graves' disease, Goodpasture's syndrome,Guillain-Barre syndrome, inflammatory bowel disease, Lupus nephritis,multiple sclerosis, myasthenia gravis, myocarditis, Parkinson disease,pemphigus, pernicious anemia, polyarteritis nodosa, polymyositis,primary biliary cirrhosis, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, sjogren's syndrome, systemic lupuserythematosus, thyroiditis, ulcerative colitis, uveitis, vitiligo,Wegener's granulomatosis, and Wilsons diseases, for example.

In an example in accordance with the principles described herein, by wayof illustration and not limitation, the receptor is a TSH receptor andthe autoantibodies are TSH autoantibodies. In another example inaccordance with the principles described herein, by way of illustrationand not limitation, the receptor is a TSH receptor chimera and theautoantibodies are TSH autoantibodies. In another example in accordancewith the principles described herein, by way of illustration and notlimitation, the receptors are TSH receptor chimeras, examples of whichare disclosed in U.S. Patent Application Publication No. 2009/0325310 A1(Loos) published Dec. 31, 2009, and the autoantibodies are TSHautoantibodies.

The term “receptor” also includes receptors bound to or attached to aconjugative moiety. Receptor conjugates comprise a receptor and aconjugative moiety bound together, optionally through a linking group,to form a single structure. The binding can be the result of eithercovalent attachment or non-covalent attachment. Covalent attachmentinvolves a direct connection, e.g., a chemical bond between a receptorand a conjugative moiety or between a receptor and a conjugative moietythrough the intermediacy of a linking group. Non-covalent attachmentinvolves specific binding between complementary specific binding pair(sbp) members that are attached to a receptor and a conjugative moietyof the conjugate.

The conjugative moiety is any entity that may be conjugated to areceptor to form a reagent that is employed in an assay for thedetection of the receptor or an entity to which the receptor binds(receptor-binding analyte). Conjugative moieties include, by way ofillustration and not limitation, supports, members of a signal producingsystem, members of binding pairs such as, for example, ligands andreceptors (e.g., biotin-streptavidin or fluorescein-anti-fluoresceinantibodies), and macromolecules that provide anchors to a drug analog,for example.

A support may be comprised of an organic or inorganic, solid or fluid,water insoluble material, which may be transparent or partiallytransparent. The support may be synthetic or naturally-occurring. Thesupport can have any of a number of shapes, such as particle, includingbead, film, membrane, tube, well, strip, rod, planar surfaces such as,e.g., plate, paper, etc., fiber, and the like. Depending on the type ofassay, the support may or may not be suspendable in the medium in whichit is employed. Examples of suspendable supports are polymeric materialssuch as latex, lipid bilayers or liposomes, oil droplets, cells andhydrogels, metallic particles, and magnetic particles, for example.Other support compositions include polymers, such as cross-linkedpolysaccharides including agarose and dextran, for example, cellulose,nitrocellulose, cellulose acetate, polyvinyl alcohol, poly (vinylchloride), polyacrylamide, polyacrylate, polymethacrylates,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, and poly(vinylbutyrate), for example, either used by themselves or in conjunction withother materials.

The support may be a particle. The particles may have an averagediameter of at least about 0.02 microns and not more than about 100microns. In some embodiments, the particles have an average diameterfrom about 0.05 microns to about 20 microns, or from about 0.3 micronsto about 10 microns. The particle may be organic or inorganic, swellableor non-swellable, porous or non-porous, preferably of a densityapproximating water, generally from about 0.7 g/mL to about 1.5 g/mL,and composed of material that can be transparent, partially transparent,or opaque. The particles can be biological materials such as cells andmicroorganisms, e.g., erythrocytes, leukocytes, lymphocytes, hybridomas,streptococcus, Staphylococcus aureus, E. coli, and viruses, for example.The particles can also be comprised of organic and inorganic polymers,liposomes, latex particles, metallic particles, magnetic or non-magneticparticles, phospholipid vesicles, chylomicrons, and lipoproteins, forexample. In some examples, the particles are latex particles or chromiumdioxide (chrome) particles. In some examples, the particles are readilydispersible in an aqueous medium and can be adsorptive orfunctionalizable so as to permit conjugation to a receptor, eitherdirectly or indirectly through a linking group.

Specific signal producing systems and labels are discussed in moredetail hereinbelow in the discussion of various assay systems to whichexamples in accordance with the principles described herein may beapplied. Briefly, a signal producing system (sps) may have one or morecomponents or members, at least one component or member being a label,which is capable of being detected directly or is detectable through aspecific binding reaction that produces a detectable signal. The signalproducing system generates a signal that relates to the presence of areceptor or a receptor-binding analyte in a sample. The signal producingsystem includes all of the reagents required to produce a measurablesignal. Other components of the signal producing system may be includedin a developer solution and can include substrates, enhancers,activators, chemiluminescent compounds, cofactors, inhibitors,scavengers, metal ions, and specific binding substances required forbinding of signal generating substances, for example. Other componentsof the signal producing system may be coenzymes, substances that reactwith enzymic products, other enzymes and catalysts, for example. Thesignal producing system provides a signal detectable by external means,by use of electromagnetic radiation, desirably by visual examination.Exemplary signal-producing systems are described in U.S. Pat. No.5,508,178 (Rose, et al.) and Ullman, et al., U.S. Pat. No. 5,185,243,columns 11-13, the relevant disclosure of which is incorporated hereinby reference.

For covalent attachment of the components of a conjugate, that is areceptor and a conjugative moiety, one or more of the componentscontains a functional group suitable for attachment to one or more ofthe other components. The functional groups suitable for attaching thecomponents may be carbonyl functionalities, both oxocarbonyl, e.g.,aldehyde, and non-oxocarbonyl (including nitrogen and sulfur analogs)e.g., carboxy, amidine, amidate, thiocarboxy and thionocarboxy.Alternative functionalities of oxo include active halogen, diazo,mercapto, olefin, particularly activated olefin, amino, phosphoro andthe like. Of particular interest are activated esters or alkylatingagents. Details of techniques for attaching molecules to one another maybe found, for example, in Matthews, et al., Anal. Biochem. (1985)151:205-209; Engelhardt, et al., European Patent Application No. 0302175and U.S. Pat. No. 3,817,837, the relevant disclosure of which isincorporated herein by reference in its entirety.

As mentioned above, covalent attachment may be achieved through theintermediacy of a linking group. The linking group may be a chain offrom 1 to about 50 or more atoms, or from 1 to about 30 or more atoms,from about 1 to about 20 atoms, from 1 to about 10 atoms, eachindependently selected from the group normally consisting of carbon,oxygen, sulfur, nitrogen, and phosphorous, usually carbon and oxygen.The number of heteroatoms in the linking group may range from 0 to about10 or more, or from 0 to about 8, from 1 to about 6, from 2 to about 4.The number of atoms in the chain is determined by counting the number ofatoms other than hydrogen or other monovalent atoms along the shortestroute between the substructures being connected. The atoms of thelinking group may be substituted with atoms other than hydrogen such ascarbon, oxygen and so forth in the form, e.g., of alkyl, aryl, aralkyl,hydroxyl, alkoxy, aryloxy, and aralkoxy, for example. As a general rule,the length of a particular linking group can be selected arbitrarily toprovide for convenience of synthesis with the proviso that there beminimal interference caused by the linking group with the ability of thecompounds to be linked together. The linking group may be aliphatic oraromatic. Functionalities present in the linking group may includeesters, thioesters, amides, thioamides, ethers, ureas, thioureas,guanidines, azo groups, thioethers, carboxylate and so forth.

As indicated above, the components, i.e., receptor and conjugativemoiety, of receptor-moiety conjugates may be attached togethernon-covalently. For example, a small organic molecule such as, but notlimited to, biotin including bis-biotin, and fluorescein, for example,may be incorporated into one of the components and the other componentmay be linked to a binding partner for the small organic molecule suchas, for example, respectively, avidin (streptavidin) oranti-fluorescein. The binding of the binding partners results in thenon-covalent attachment of the components to one another.

Methods in accordance with the principles described herein comprisecombining in a liquid medium the receptor and effective amounts of achelating agent and a C2-C6 polyol. In some examples in accordance withthe principles described herein, the chelating agent may be, but is notlimited to, N-(2-hydroxyethyl)-ethylenediamine-N,N′,N′-triacetic acid(HEDTA), ethylenediamine-tetraacetic acid (EDTA),trans-1,2-diamino-cyclohexan-N,N,N′,N′-tetraacetic acid (CDTA), ethyleneglycol-O,O′-bis-(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),diethylenetriamine-pentaacetic acid (DTPA), nitrilotriacetic acid (NTA),nitrilo-2,2′,2″-triacetic acid,diethylenetriamine-N,N,N′,N′,N″-pentaacetic acid,triethylenetetramin-N,N,N,N″,N″,N″-hexaacetic acid (TTHA), methylamine,histidine, malate and phytochelatin, hemoglobin, chlorophyll,siderophore, pyocyanin, pyoverdin, Enterobactin, peptides and sugars,humic acid, citric acid, water softeners, phosphonates, tetracycline,gadolinium, organophosphorus compound2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, pentetic acid;N,N-Bis(2-(bis-(carboxymethyl)amino)ethyl)-glycine,N,N-bis(carboxymethyl)glycine, triglycollamic acid;[(Carboxymethyl)imino]bis-(ethylenenitrilo)]-tetraacetic acid), TriloneA, α,α′,α″-trimethylaminetricarboxylic acid, tri(carboxymethyl)amine,aminotriacetic acid, Titriplex and Hampshire NTA acid, for example, andappropriate salts of any of the above.

In some examples in accordance with the principles described herein, thechelating agent comprises a triacetic acid moiety or a salt thereof, atetraacetic acid moiety or a salt thereof, a pentaacetic acid moiety ora salt thereof, or a hexaacetic acid moiety or a salt thereof. In someexamples, the chelating agent is selected from the group consisting ofN-(2-hydroxyethyl)-ethylenediamine-N, N′,N′-triacetic acid and itssalts, ethylene diamine tetraacetic acid and its salts, and ethyleneglycol tetraacetic acid and its salts. In some examples, the chelatingagent is a citric acid or a salt thereof.

In some examples in accordance with the principles described herein, thenumber of carbon atoms in the polyol is 2 to 8, or 2 to 7, or 2 to 6, or2 to 5, or 2 to 4, or 2 to 3, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to5, or 3 to 4, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, for example,and the number of hydroxyl groups is about 1 per carbon atom, or 1 per 2carbon atoms, or 1 per 3 carbon atoms, or 2 per 2 carbon atom, or 2 per3 carbon atoms, for example, with the total number of hydroxyl groups inthe polyol being about 2, or about 3, or about 4, or about 5, or about6, or about 7, or about 8, for example. In some examples, by way ofillustration and not limitation, the polyol is a C₂, or C₃, or C₄, orC₅, or C₆ polyol comprising 2 hydroxy groups or 3 hydroxy groups, or 4hydroxyl groups, or 5 hydroxyl groups, or 6 hydroxyl groups such as, byway of illustration and not limitation, ethylene glycol, propyleneglycol, glycerol, erythritol, xylitol, ribitol, and sorbitol, forexample. The polyol may be a single compound or a combination of two ormore polyols having the aforementioned properties. In some examples inaccordance with the principles described herein, the polyol is glycerol.

The chelating agent and the polyol are present in the combination inamounts that are effective to stabilize the receptor and/or to enhancethe sensitivity of the receptor. The amount of the chelating agent andthe amount of the polyol are dependent on one or more of the nature andamount of the receptor, the nature of the chelating agent, the nature ofthe polyol, and the nature of the liquid formulation, for example. Insome examples in accordance with the principles described herein, aneffective amount of the chelating agent in the combination may be, forexample, about 0.1 mM to about 20 mM, or about 1 mM to about 20 mM, orabout 5 mM to about 20 mM, or about 10 mM to about 20 mM, or about 10 mMto about 15 mM, or about 15 mM to about 20 mM or about 1 to about 5 mM.

In some examples in accordance with the principles described herein, aneffective amount of the polyol in the combination may be, for example,about 5% to about 50%, or about 5% to about 40%, or about 5% to about30%, or about 5% to about 20%, or about 5% to about 10%, or about 10% toabout 50%, or about 10% to about 40%, or about 10% to about 30%, orabout 10% to about 20%, for example. The above percentages are by weightof the polyol in a liquid medium.

Some examples in accordance with the principles described herein aredirected to compositions comprising an aqueous medium, a receptor, achelating agent in an amount of about 0.1 mM to about 20 mM, and a C2-C6polyol in an amount of about 5% to about 50% by weight.

In some examples, the liquid medium or liquid solution is an aqueousmedium, which may be solely water or may include about 0.1% to about80%, or 0.1% to about 60%, or about 0.1 to about 40%, or about 0.1% toabout 30%, or about 0.1% to about 20%, or about 0.1% to about 10%, orabout 0.1% to about 5%, or about 1% to about 80%, or 1% to about 60%, orabout 1% to about 40%, or about 1% to about 30%, or about 1% to about20%, or about 1% to about 10%, or about 1% to about 5%, or about 5% toabout 80%, or 5% to about 60%, or about 5% to about 40%, or about 5% toabout 30%, or about 5% to about 20%, or about 5% to about 10%, forexample, of a cosolvent. The above percentages are by volume of themedium. The cosolvent may be, for example, but not limited to, anorganic solvent such as, for example, an alcohol, an ester, an ether, anamide or an amine.

The pH for the medium will usually be in the range of about 4 to about11, or in the range of about 5 to about 10, or in the range of about 6.5to about 9.5, or in the range of about 6.5 to about 7.5, or about 7, forexample. Various buffers may be used to achieve the desired pH andmaintain the pH of the medium. Illustrative buffers include borate,phosphate, carbonate, tris, and barbital, for example. The particularbuffer employed is not critical, but in an individual composition one oranother buffer may be preferred. Various ancillary materials also may beincluded in the medium. For example, in addition to buffers the mediummay contain preservatives, non-specific binding blockers andheterophilic interference blockers to prevent false results, andsurfactants, for example.

The liquid solution of the receptor, the chelating agent and the C2-C6polyol may be maintained at a temperature of about 2° C. to about 40°C., or about 2° C. to about 30° C., or about 2° C. to about 20° C., orabout 5° C. to about 40° C., or about 5° C. to about 30° C., or about 5°C. to about 20° C., or about 5° C. to about 10° C., or about 10° C. toabout 40° C., or about 10° C. to about 30° C., or about 10° C. to about20° C., or about 15° C. to about 40° C., or about 15° C. to about 30°C., or about 15° C. to about 20° C., or about 20° C. to about 25° C., orabout ambient temperature, for example. The liquid solution of thereceptor maintains good stability for at least about 1 week, or at leastabout 1 month, or at least about 6 months, or at least about 1 year, orat least about 1.5 years, or at least about 2 years, for example. Thephrase “good stability” as used herein means that the receptor does notlose more than about 40%, or more than about 30%, or more than about20%, or more than about 10%, or more than about 5% of its activity overa period of 12 months or that the receptor has a half-life of at leastabout 12 months, or at least about 15 months, or at least about 20months during storage at a temperature of about 2° C. to about 10° C.

The compositions in accordance with the principles described herein maybe employed in methods of detecting in a sample a receptor-bindinganalyte, which refers to a molecule that binds to the receptor. Thesample to be analyzed is one that is suspected of containing one or morereceptor-binding analytes. The samples are preferably from humans oranimals and include, but are not limited to, biological fluids such aswhole blood, serum, plasma, sputum, lymphatic fluid, semen, vaginalmucus, feces, urine, spinal fluid, saliva, stool, cerebral spinal fluid,tears, and mucus, for example, and biological tissue such as hair, skin,sections or excised tissues from organs or other body parts, forexample. In many instances, the sample is whole blood, plasma or serumand, in a particular example, the sample is serum. The sample may or maynot be pretreated to remove endogenous binding moieties that bind to thereceptor-binding analyte or to release the receptor-binding analyte fromendogenous binding substances.

The sample can be prepared in any convenient medium that does notinterfere with an assay; an aqueous medium generally is employed. Thenature of the medium is discussed in more detail below. Depending on thenature of the sample, one or more pretreatments may be carried out onthe sample such as, by way of illustration and not limitation,pretreatment with a hemolytic agent, pretreatment with a releasingagent, pretreatment with a displacer that displaces boundreceptor-binding analyte from its endogenous binding partners such asproteins, and hemolytic detergent(s) that assist in lysing blood cellsto release a receptor-binding analyte from blood cells, for example. Anyof the above agents are present in a concentration or amount sufficientto achieve the desired effect or function, such as, for example,hemolysis or release of receptor-binding analyte from endogenous bindingsubstances.

Following the above pretreatment, if any, reagents for determining oneor both of the presence and amount of the receptor-binding analyte inthe sample are added to the medium. The nature of the reagents isdependent on the particular type of assay to be performed. In general,the assay is a method for the determination or measuring of one or bothof the presence and amount of a receptor-binding analyte. Various assaymethods are discussed below by way of illustration and not limitation.

In some examples in accordance with the principles described herein, theassay reagents include at least one receptor for the receptor-bindinganalyte. The receptor is present in a liquid composition or liquidsolution in accordance with the principles described herein and aportion of the liquid composition is combined in the assay medium. Asmentioned above, receptors treated with a combination of a polyol and achelating agent in accordance with the principles described herein alsoexhibit enhanced sensitivity over receptors that were not treated inthis manner. The phrase “enhanced sensitivity” means that thesensitivity achieved in an assay for a receptor-binding analyte thatemploys a receptor treated in accordance with the principles describedherein is at least about 10% greater, or at least about 25% greater, orat least about 50% greater, or at least about 75% greater than thesensitivity of an assay using a receptor not treated in accordance withthe principles described herein.

In addition to one or more receptors, assay reagents may include one ormore antibodies specific for a receptor, or for a receptor-bindinganalyte, another antibody, or a small molecule, for example. Antibodiescan be monoclonal or polyclonal. Such antibodies can be prepared bytechniques that are well known in the art such as immunization of a hostand collection of sera (polyclonal) or by preparing continuous hybridcell lines and collecting the secreted protein (monoclonal) or bycloning and expressing nucleotide sequences or mutagenized versionsthereof coding at least for the amino acid sequences required forspecific binding of natural antibodies. Antibodies may include acomplete immunoglobulin or fragment thereof, which immunoglobulinsinclude the various classes and isotypes, such as IgA, IgD, IgE, IgG1,IgG2a, IgG2b and IgG3, and IgM, for example. Fragments thereof mayinclude Fab, Fv and F(ab′)₂, and Fab′, for example. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

Other reagents are included in an assay medium depending on the natureof the assay to be conducted. Such assays usually involve reactionsbetween binding partners such as a receptor and a receptor-bindinganalyte and may also include binding between an antibody and acorresponding binding partner such as, for example, a receptor,depending on the particular assay format chosen.

As discussed above, specific binding involves the specific recognitionof one of two different molecules for the other compared tosubstantially less recognition of other molecules. On the other hand,non-specific binding involves non-covalent binding between moleculesthat is relatively independent of specific surface structures.Non-specific binding may result from several factors includinghydrophobic interactions between molecules. In some examples, thebinding partners are antibodies.

General Discussion of Assays

The present disclosure has application to many types of assays that maybe employed to determine one or both of the presence and amount of oneor more analytes that include, for example, receptor-binding analytes,in a sample suspected of containing such analyte. The receptor reagentsin accordance with the principles described herein may be employed inmany assay formats that are designed for antibody reagents. The assaysmay involve labeled or non-labeled reagents. Assays involvingnon-labeled reagents usually comprise the formation of relatively largecomplexes involving one or more receptors. Such assays include, forexample, immunoprecipitin and agglutination methods and correspondinglight scattering techniques such as, e.g., nephelometry andturbidimetry, for the detection of receptor complexes. For non-labeledassays, a composition in accordance with the principles described hereinmay be employed where the composition comprises a conjugate of areceptor-binding analyte and a support such as, for example, a particle.In one example, the receptor-binding analyte in a sample competes withthe receptor-binding analyte-support conjugate such that the more theamount of receptor-binding analyte in a sample, the less will be theamount of a precipitate formed by agglutination. In some examples thereceptor-binding analyte is an antibody such as, for example, anautoantibody that may be present in a sample and a conjugate inaccordance with the principles described herein having areceptor-binding analyte bound to a particulate support is employed. Thepresence of the antibody in the sample results in the agglutination ofthe conjugate reagent.

Labeled immunoassays include enzyme immunoassays, fluorescencepolarization immunoassays, radioimmunoassay, inhibition assays, andinduced luminescence, fluorescent oxygen channeling assay, for example.In one example of a labeled assay approach, a conjugate having areceptor-binding analyte bound to a label such as, for example, anenzyme, can compete with receptor-binding analyte in a sample such thatthe greater the amount of receptor-binding analyte in the sample, theless will be the amount of signal from the label. In another example ofa labeled assay approach, a conjugate having a receptor bound to a labelsuch as, for example, an enzyme, can be employed to bind withreceptor-binding analyte in a sample and a second receptor for thereceptor-binding analyte is employed to form a sandwich complex. Anamount of signal from the label is detected and related to the amount ofthe receptor-binding analyte in the sample such that the greater theamount of receptor-binding analyte in the sample, the greater will bethe amount of signal from the label.

As mentioned above, in many of the assays discussed herein, a label isemployed and is in many examples part of a receptor-binding analyteconjugate. On the other hand the label may be part of a reagentindependent of the receptor-binding analyte conjugate. The label isusually part of a signal producing system (“sps”). The nature of thelabel is dependent on the particular assay format. As discussed above,the signal producing system usually includes one or more components, atleast one component being a detectable label, which generates adetectable signal that relates to the amount of bound and/or unboundlabel, i.e. the amount of label bound or not bound to thereceptor-binding analyte being detected or to an agent that reflects theamount of the receptor-binding analyte to be detected.

The label is any molecule that produces or can be induced to produce asignal. The label can be a poly(amino acid), or protein, ornon-poly(amino acid), isotopic or non-isotopic, usually non-isotopic,and can be a catalyst, such as an enzyme, a polynucleotide coding for acatalyst, promoter, dye, fluorescent molecule, chemiluminescentmolecule, coenzyme, enzyme substrate, radioactive group, a small organicmolecule, amplifiable polynucleotide sequence, a particle such as latexor carbon particle, metal sol, crystallite, liposome, cell, etc., whichmay or may not be further labeled with a dye, catalyst or otherdetectable group, for example. In some examples, the labels areradioisotopic, luminescent, particulate or enzymic and may be, forexample, a fluorescer, radiolabel, enzyme, chemiluminescer orphotosensitizer. Thus, for example, for the above labels, the signal isdetected and/or measured by detecting enzyme activity, luminescence,light absorbance or radioactivity, as the case may be.

The term “non-poly(amino acid) labels” refers to those labels that arenot proteins. A non-poly(amino acid) label may be a member of a signalproducing system. The non-poly(amino acid) label is capable of beingdetected directly or is detectable through a specific binding reactionthat produces a detectable signal. The non-poly(amino acid) labelsgenerally are radioisotopic, luminescent (such as, e.g., acridiniumesters), particulate (such as, e.g., magnetic particles that can beseparated bound from un-bound, latex particles that can be measured byturbidity and nephelometry, and chemiluminescence beads (e.g., LOCIchemibeads), for example. The label can be isotopic or non-isotopic,usually non-isotopic, and can be a polynucleotide coding for a catalyst,promoter, dye, fluorescent molecule, chemiluminescent molecule,coenzyme, enzyme substrate, radioactive group, a small organic molecule,amplifiable polynucleotide sequence, a particle such as, e.g., latex orcarbon particle, metal sol, crystallite, liposome, or cell, which may ormay not be further labeled with a dye, catalyst or other detectablegroup, for example. Poly(amino acid) labels include, by way ofillustration and not limitation, peptides and proteins such as e.g.,enzymes, for example.

Examples of labels include, by way of illustration and not limitation,enzymes such as alkaline phosphatase (“AP”), glucose-6-phosphatedehydrogenase (“G6PDH”) and horseradish peroxidase; ribozyme; asubstrate for a replicase such as QB replicase; promoters; dyes;fluorescers, such as fluorescein, isothiocyanate, rhodamine compounds,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, andfluorescamine; complexes such as those prepared from CdSe and ZnSpresent in semiconductor nanocrystals known as Quantum dots;chemiluminescers such as isoluminol; sensitizers; coenzymes; enzymesubstrates; radiolabels such as ¹²⁵I, ¹³¹I, ¹⁴C, ³H, ⁵⁷Co and ⁷⁵Se;particles such as latex particles, carbon particles, metal particlesincluding magnetic particles, e.g., chromium dioxide (CrO₂) particles,and the like; metal sol; crystallite; liposomes; cells, etc., which maybe further labeled with a dye, catalyst or other detectable group.Suitable enzymes and coenzymes are disclosed in Litman, et al., U.S.Pat. No. 4,275,149, columns 19-28, and Boguslaski, et al., U.S. Pat. No.4,318,980, columns 10-14; suitable fluorescers and chemiluminescers aredisclosed in Litman, et al., U.S. Pat. No. 4,275,149, at columns 30 and31; which are incorporated herein by reference.

The label can directly produce a signal and, therefore, additionalcomponents are not required to produce a signal. Numerous organicmolecules, for example fluorescers, are able to absorb ultraviolet andvisible light, where the light absorption transfers energy to thesemolecules and elevates them to an excited energy state. This absorbedenergy is then dissipated by emission of light at a second wavelength.Other labels that directly produce a signal include radioactive isotopesand dyes.

Alternately, the label may need other components to produce a signal,and the signal producing system would then include all the componentsrequired to produce a measurable signal. Such other components mayinclude substrates, coenzymes, enhancers, additional enzymes, substancesthat react with enzymic products, catalysts, activators, cofactors,inhibitors, scavengers, metal ions, and a specific binding substancerequired for binding of signal generating substances.

In some examples, enzymes of interest as label proteins are redoxenzymes, particularly dehydrogenases such as glucose-6-phosphatedehydrogenase, lactate dehydrogenase, etc., and enzymes that involve theproduction of hydrogen peroxide and the use of the hydrogen peroxide tooxidize a dye precursor to a dye. Particular combinations includesaccharide oxidases, e.g., glucose and galactose oxidase, orheterocyclic oxidases, such as uricase and xanthine oxidase, coupledwith an enzyme which employs the hydrogen peroxide to oxidize a dyeprecursor, that is, a peroxidase such as horse radish peroxidase,lactoperoxidase, or microperoxidase. Additional enzyme combinations areknown in the art. When a single enzyme is used as a label, other enzymesmay find use such as hydrolases, transferases, and oxidoreductases,preferably hydrolases such as alkaline phosphatase andbeta-galactosidase. Alternatively, luciferases may be used such asfirefly luciferase and bacterial luciferase. Illustrative co-enzymesthat find use include NAD[H], NADP[H], pyridoxal phosphate, FAD[H],FMN[H], etc., usually coenzymes involving cycling reactions. See, forexample, U.S. Pat. No. 4,318,980, the disclosure of which isincorporated herein by reference.

One general group of assays that may be employed includes assays using alimited concentration of receptor. Another group of assays involves theuse of an excess of one or more of the principal reagents such as, forexample, an excess of a receptor for the receptor-binding analyte.Another group of assays are separation-free homogeneous assays in whichthe labeled reagents modulate the label signal upon receptor-bindinganalyte-receptor binding reactions. Another group of assays includeslabeled receptor reagent limited competitive assays for areceptor-binding analyte. In this type of assay, a receptor-supportconjugate in accordance with the principles described herein is presentin a constant, limited amount. The partition of a label between theimmobilized receptor-binding analyte and free receptor-binding analytedepends on the concentration of receptor-binding analyte in the sample.

The assays can be performed either without separation (homogeneous) orwith separation (heterogeneous) of any of the assay components orproducts. Receptors in accordance with the principles described hereincan be employed in many immunoassay formats. In a homogeneous assayafter all of the reagents have been combined, the signal is determinedand related to the amount of receptor-binding analyte in the sample.Homogeneous immunoassays are exemplified by the EMIT® assay (SyvaCompany, San Jose, Calif.) disclosed in Rubenstein, et al., U.S. Pat.No. 3,817,837, column 3, line 6 to column 6, line 64; immunofluorescencemethods such as those disclosed in Ullman, et al., U.S. Pat. No.3,996,345, column 17, line 59, to column 23, line 25; enzyme channelingimmunoassays (“ECIA”) such as those disclosed in Maggio, et al., U.S.Pat. No. 4,233,402, column 6, line 25 to column 9, line 63; thefluorescence polarization immunoassay (“FPIA”) as disclosed, forexample, in, among others, U.S. Pat. No. 5,354,693; and so forth.

Other enzyme immunoassays are the enzyme modulate mediated immunoassay(“EMMIA”) discussed by Ngo and Lenhoff, FEBS Lett. (1980) 116:285-288;the substrate labeled fluorescence immunoassay (“SLFIA”) disclosed byOellerich, J. Clin. Chem. Clin. Biochem. (1984) 22:895-904; the combinedenzyme donor immunoassays (“CEDIA”) disclosed by Khanna, et al., Clin.Chem. Acta (1989) 185:231-240; homogeneous particle labeled immunoassayssuch as particle enhanced turbidimetric inhibition immunoassays(“PETINIA”), particle enhanced turbidimetric immunoassay (“PETIA”),etc.; and the like.

Other assays include the sol particle immunoassay (“SPIA”), the dispersedye immunoassay (“DIA”); the metalloimmunoassay (“MIA”); the enzymemembrane immunoassays (“EMIA”); luminoimmunoassays (“LIA”); and soforth. Other types of assays include immunosensor assays involving themonitoring of the changes in the optical, acoustic and electricalproperties of an antibody-immobilized surface upon the binding of areceptor-binding analyte. Such assays include, for example, opticalimmunosensor assays, acoustic immunosensor assays, semiconductorimmunosensor assays, electrochemical transducer immunosensor assays,potentiometric immunosensor assays, and amperometric electrode assays.

In one example of an enzyme assay for a receptor-binding analyte, asample suspected of containing the receptor-binding analyte is combinedin an aqueous medium either simultaneously or sequentially with areceptor capable of recognizing the receptor-binding analyte and areagent that comprises a conjugate of the receptor-binding analyte andan enzyme. The receptor is added from a liquid solution of the receptorstabilized in accordance with the principles described herein. Asubstrate for the enzyme is added, which results in the formation of achromogenic or fluorogenic product upon enzyme-catalyzed reaction.Examples, by way of illustration and not limitation, of enzymes areglucose-6-phosphate dehydrogenase and alkaline phosphatase but otherenzymes may be employed. The receptor-binding analyte and thereceptor-binding analyte moiety of the enzyme conjugate compete forbinding sites on the receptor. The enzyme activity in the medium is thendetermined, usually by spectrophotometric means, and is compared to theenzyme activity determined when calibrators or reference samples aretested, in which a known amount of the receptor-binding analyte ispresent. Typically, the calibrators are tested in a manner similar tothe testing of the sample suspected of containing the receptor-bindinganalytes. The calibrators contain differing, but known concentrations ofthe receptor-binding analyte to be determined. In most examples, theconcentration ranges present in the calibrators span the range ofsuspected receptor-binding analyte concentrations in unknown samples.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive assay formats are disclosed in Davalian, et al., U.S.Pat. No. 5,089,390, column 14, line 25 to column 15, line 9, whichdisclosure is incorporated herein by reference. In one type ofcompetitive assay, a support, as discussed herein, having a receptor forthe receptor-binding analyte bound thereto is contacted with a mediumcontaining the sample suspected of containing the receptor-bindinganalyte and a reagent that is a conjugate of the receptor-bindinganalyte and an enzyme. The support reagent with attached receptor is ina liquid solution in accordance with the principles described herein.After separating the support and the medium, the enzyme activity of thesupport or the medium is determined by conventional techniques andrelated to one or both of the presence and amount of thereceptor-binding analyte in the sample. In certain examples a secondenzyme may be employed in addition to the enzyme of the enzymeconjugate. The enzymes of the pair of enzymes are related in that aproduct of the first enzyme serves as a substrate for the second enzyme.

Another example of an assay format is a capture assay. In this assayformat, the receptor for the receptor-binding analyte is covalentlybound to a particle such as, for example, a magnetic particle.Alternatively, the receptor may be bound to the particle non-covalentlysuch as, for example, by an antibody for the receptor where the antibodyis covalently attached to the particle. This receptor-particle reagentis stored in a liquid solution in accordance with the principlesdescribed herein prior to its use in the assay. The sample is incubatedwith the receptor-particle reagent to allow the receptor-binding analytein the sample to bind to the receptor for the receptor-binding analyte.Optionally, the particles are separated from the assay medium andwashed. A reagent that comprises a conjugate of a second receptor forthe receptor-binding analyte, which receptor is bound to an enzyme, isincubated with the particles. This second receptor-particle reagent maybe stored in a liquid solution in accordance with the principlesdescribed herein prior to its use in the assay. After washing, theamount of enzyme that is bound to the separated particles is measuredand is directly related to one or both of the presence and amount of thereceptor-binding analyte in the sample.

In some embodiments multi-analyte assays may be utilized where thereceptor-binding analyte may be the subject of detection along with oneor more other analytes such as other receptor-binding analytes, forexample. Such multi-analyte systems are described, for example, in Loor,et al., J. Anal. Toxicol. 12: 299 (1988).

The assays discussed above are normally carried out in an aqueousbuffered medium at a moderate pH, generally that which provides optimumassay sensitivity. The pH for the assay medium may be in the range ofabout 4 to about 11, or in the range of about 5 to about 10, or in therange of about 6.5 to about 9.5, for example. The pH will usually be acompromise between optimum binding of the binding members of anyspecific binding pairs, the pH optimum for other reagents of the assaysuch as members of the signal producing system, for example.

Various buffers may be used to achieve the desired pH and maintain thepH during the determination. Illustrative buffers include borate,phosphate, carbonate, tris, barbital and the like. The particular bufferemployed is not critical, but in an individual assay one or anotherbuffer may be preferred. Various ancillary materials may be employed inthe above methods. For example, in addition to buffers the medium maycomprise stabilizers for the medium and for the reagents employed.Frequently, in addition to these additives, proteins may be included,such as albumins; quaternary ammonium salts; polyanions such as dextransulfate; and binding enhancers, for example.

One or more incubation periods may be applied to the assay medium at oneor more intervals including any intervals between additions of variousreagents mentioned above. The medium is usually incubated at atemperature and for a time sufficient for binding of various componentsof the reagents to occur. Moderate temperatures are normally employedfor carrying out the method and usually constant temperature,preferably, room temperature, during the period of the measurement.Incubation temperatures normally range from about 5° C. to about 99° C.or from about 15° C. to about 70° C. or from about 20° C. to about 45°C., for example. The time period for the incubation is about 0.2 secondsto about 24 hours, or about 1 second to about 6 hours, or about 2seconds to about 1 hour, or about 1 to about 15 minutes. The time perioddepends on the temperature of the medium and the rate of binding of thevarious reagents, which is determined by the association rate constant,the concentration, the binding constant and dissociation rate constant.Temperatures during measurements will generally range from about 10° C.to about 50° C. or from about 15° C. to about 40° C., for example.

The concentration of receptor-binding analyte that may be assayedgenerally varies from about 10⁻⁵ to about 10⁻¹⁷ M, more usually fromabout 10⁻⁶ to about 10⁻¹⁴ M. Considerations, such as whether the assayis qualitative, semi-quantitative or quantitative (relative to theamount of receptor-binding analyte present in the sample), theparticular detection technique and the concentration of the analytenormally determine the concentrations of the various reagents.

The concentrations of the various reagents in the assay medium willgenerally be determined by the concentration range of interest of thereceptor-binding analyte and the nature of the assay, for example.However, the final concentration of each of the reagents is normallydetermined empirically to optimize the sensitivity of the assay over therange. That is, a variation in concentration of receptor-binding analytethat is of significance should provide an accurately measurable signaldifference. Considerations such as the nature of the signal producingsystem and the nature of the receptor-binding analytes normallydetermine the concentrations of the various reagents.

While the order of addition may be varied widely, there will be certainpreferences depending on the nature of the assay. The simplest order ofaddition is to add all the materials simultaneously and determine theeffect that the assay medium has on the signal as in a homogeneousassay. Alternatively, the reagents can be combined sequentially. In someexamples, an incubation step may be involved subsequent to each additionas discussed above.

Examination Step

In a next step of an assay method, the medium is examined for thepresence of a complex comprising the receptor-binding analyte and thereceptor for the receptor-binding analyte. The presence and/or amount ofthe complex indicates the presence and/or amount of the receptor-bindinganalyte in the sample.

The phrase “measuring the amount of a receptor-binding analyte” refersto the quantitative, semiquantitative and qualitative determination ofthe receptor-binding analyte. Methods that are quantitative,semiquantitative and qualitative, as well as all other methods fordetermining the receptor-binding analyte, are considered to be methodsof measuring the amount of the receptor-binding analyte. For example, amethod, which merely detects the presence or absence of thereceptor-binding analyte in a sample suspected of containing thereceptor-binding analyte, is considered to be included within the scopeof the present invention. The terms “detecting” and “determining,” aswell as other common synonyms for measuring, are contemplated within thescope of the present invention.

In many embodiments the examination of the medium involves detection ofa signal from the medium. One or both of the presence and amount of thesignal are related to the presence and/or amount of the receptor-bindinganalyte in the sample. The particular mode of detection depends on thenature of the signal producing system. As discussed above, there arenumerous methods by which a label of a signal producing system canproduce a signal detectable by external means, desirably by visualexamination, and include, for example, electromagnetic radiation,electrochemistry, heat, radioactivity detection, and chemical reagents.

Activation of a signal producing system depends on the nature of thesignal producing system members. For those members of a signal producingsystem that are activated with light, the member is irradiated withlight. Other activation methods will be suggested to those skilled inthe art in view of the disclosures herein. For some signal producingsystems, no agent for activation is necessary such as those systems thatinvolve a label that is a radioactive label or an enzyme, for example.For enzyme systems, addition of a substrate and/or a cofactor may benecessary.

The examination for the presence and/or amount of the signal alsoincludes the detection of the signal, which is generally merely a stepin which the signal is read. The signal is normally read using aninstrument, the nature of which depends on the nature of the signal. Theinstrument may be a spectrophotometer, fluorometer, absorptionspectrometer, luminometer, chemiluminometer, actinometer, orphotographic instrument, for example. The presence and amount of signaldetected is related to the presence and amount of the receptor-bindinganalyte present in a sample. Temperatures during measurements generallyrange from about 10° C. to about 70° C. or from about 20° C. to about45° C., or about 20° C. to about 25° C., for example. In one approachstandard curves are formed using known concentrations of thereceptor-binding analytes to be screened. As discussed above,calibrators and other controls may also be used.

Specific Embodiments of Assays

The following examples describe specific examples in accordance with theprinciples described herein by way of illustration and not limitationand are intended merely to describe, and not to limit, the scope of thepresent disclosure and the appended claims. Selection of TSHautoantibodies as the receptor-binding analyte and first and second TSHreceptor chimeras is also by way of illustration and not limitationsince the examples in accordance with the principles described hereinhave general application to detection of receptor-binding analytes ingeneral and the use of receptors of all different types as discussedabove.

In one example in accordance with the principles described herein, theassay employed is an induced luminescence assay, which is described inU.S. Pat. No. 5,340,716 (Ullman, et al.) and which disclosure isincorporated herein by reference. The reagents include two latex beadreagents and a biotinylated second TSH receptor chimera for the TSHautoantibodies. The first bead reagent is a conjugate wherein one latexbead that contains a chemiluminescent dye is bound either covalently ornon-covalently to a first TSH receptor chimera for the TSHautoantibodies. In some examples, both the first TSH receptor chimerareagent and the second TSH receptor chimera reagent are separatelystored in liquid solutions in accordance with the principles describedherein. The second bead reagent is coated with streptavidin and containsa photosensitizer dye. In a first step, sample suspected of containingTSH autoantibodies is incubated with biotinylated second TSH receptorchimera for TSH autoantibodies, which allows TSH autoantibodies from thesample to saturate a fraction of the biotinylated TSH second receptorchimera where the fraction is directly related to the TSH autoantibodiesconcentration in the assay medium. In a second step, the first beadreagent, which as mentioned above is attached to a first TSH receptorchimera for the TSH autoantibodies is added and leads to the formationof bead-biotinylated TSH second receptor chimera complexes with thenon-saturated fraction of the biotinylated second TSH receptor chimera.The second bead reagent is then added and binds to the biotin to formbead pair complexes. When illuminated by light at 680 nm, the secondbead reagent converts dissolved oxygen in the reaction solution into themore energetic singlet oxygen form. In the bead pairs, the singletoxygen diffuses into the first bead reagent thereby triggering achemiluminescent reaction. The resulting chemiluminescent signal ismeasured at 612 nm and is a function of the concentration of TSHautoantibodies in the sample. The amount of this signal is related tothe presence and or amount of TSH autoantibodies in the sample.

In another example in accordance with the present disclosure, a testsample suspected of containing TSH autoantibodies is mixed with areagent (Reagent A) and a dry bead having an antibody for the TSHreceptor chimera attached to the bead and a first TSH receptor chimerabound to the antibody of the bead. The sample, bead and Reagent A areincubated for a period of time and under conditions sufficient for someof the TSH autoantibodies in the sample to become bound to the TSHreceptor chimera. The liquid reagent is separated from the bead bycentrifugation and then washed. The washed bead is incubated withReagent B containing a second TSH receptor chimera bound to alkalinephosphatase (detection receptor) for a period of time and underconditions for the detection receptor to bind to the TSH autoantibodiesbound to the first TSH receptor chimera of the bead. The bead isseparated from the assay medium and washed. Then, the bead is incubatedwith a substrate solution comprising a substrate for alkalinephosphatase and the medium is then examined for one or both of thepresence and amount of signal, which is measured with a luminometer overtime. The presence and/or amount of signal is related to the presenceand/or amount of TSH autoantibodies in the original sample.

Kits

The reagents for conducting a particular assay may be present in a kituseful for conveniently performing an assay for the determination of areceptor-binding analyte. In one example in accordance with theprinciples described herein, a kit comprises in packaged combination areceptor for a receptor-binding analyte and other reagents forperforming an assay for detection of the receptor-binding analyte wherethe nature of such reagents depends upon the particular assay format.The receptor reagent is present in a liquid solution that comprises achelating agent and a polyol in accordance with the principles describedherein. The kit can also comprise a detection receptor agent thatcomprises a label. The detection receptor agent is present in a liquidsolution that comprises a chelating agent and a polyol in accordancewith the principles described herein. The reagents may each be inseparate containers or various reagents can be combined in one or morecontainers depending on the cross-reactivity and stability of thereagents. The kit can further include other separately packaged reagentsfor conducting an assay such as additional binding reagents andancillary reagents such as an enzyme substrate reagent, for example.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the assay method andfurther to optimize substantially the sensitivity of the assay. Reagentsother than those that contain a receptor in accordance with theprinciples described herein can be provided under appropriatecircumstances as a dry powder, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing a method or assayin accordance with the present invention. The kit can further include awritten description of a method in accordance with the present inventionas described above.

Definitions

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited.

The phrase “about” as used herein means that the number recited maydiffer by plus or minus 10%; for example, “about 5” means a range of 4.5to 5.5.

The designations “first” and “second” are used solely for the purpose ofdifferentiating between two items such as, for example, “first receptor”and “second receptor,” and are not meant to imply any sequence or orderor importance to one item over another.

EXAMPLES

The following examples are by way of illustration and not limitation onthe scope of the present disclosure and the appended claims. Numerousmodifications and alternative compositions, methods, and systems may bedevised without departing from the spirit and scope of the presentdisclosure. Unless otherwise indicated, materials in the experimentsbelow may be purchased from Sigma-Aldrich Chemical Company, St. LouisMo. Parts and percentages are by weight unless indicated otherwise.

The proceeding examples describe an assay and utilize a kit for thequantitative detection of Thyroid Stimulating Immunoglobulins (TSI),which are autoantibodies to TSH receptor. The measurement of TSI inserum samples is used as an aid in the assessment of patients suspectedof having Graves' disease. The kit consists of a dry bead, a sampleincubation buffer (Reagent A), and a detection buffer (Reagent B)containing a TSH receptor chimera (as disclosed in U.S. PatentApplication Publication No. 2009/0325310 A1 published Dec. 31, 2009)conjugated to AP.

Abbreviations

L=liter(s)

IU=international units

KCPS=kilocount(s) per second

Citrate buffer=100 mM sodium citrate, 100 mM NaCl, 10 mM magnesiumacetate, 1 mM zinc chloride, 0.5% BSA, 0.1% mouse IgG, 0.2% PLURONIC®F68, 10% glycerol, pH 6.0-6.8

BSA=bovine serum albumin

IgG=immunoglobulin

MES: 2-(N-morpholino)ethanesulfonic acid

MES buffer=100 mM MES, 100 mM NaCl, 10 mM magnesium acetate, 1 mM zincchloride, 0.5% BSA, 0.1% mouse IgG, 0.2% PLURONIC® F68, 10% glycerol, pH6.0-6.8

PIPES buffer=100 mM PIPES, 100 mM NaCl, 10 mM magnesium acetate, 1 mMzinc chloride, 0.5% BSA, 0.1% mouse IgG, 0.2% PLURONIC® F68, 10%glycerol, pH 6.0-6.8

Example 1 Coating TSH Receptor on Beads

Polystyrene beads are incubated overnight with a buffer solution (100 mMsodium carbonate, 150 mM sodium chloride, 0.1% sodium azide, pH 9) of amonoclonal antibody (MAb) to TSH receptor. Following washes with thesame buffer to remove excess MAb, beads are incubated for two hours witha buffer solution (100 mM PIPES, 2 mg/mL BSA, pH 6.8) of a TSH receptorchimera. Following washes with the same buffer to remove excess TSHreceptor, the beads are incubated with 100 mM PIPES, 25 mg/mL BSA, 5%sucrose, pH 6.8 for one hour and then dried in a vacuum oven.

Example 2 Effect of HEDTA in TSH Receptor Buffer

Serum samples with varying amounts of native TSI were incubated for 30minutes with a polystyrene bead having human TSH receptor chimeraimmobilized thereon as described in Example 1. Following centrifugalwashes of the beads, a second TSH receptor chimera conjugated toalkaline phosphatase (AP) in a detection buffer (100 mM PIPES, 100 mMKCl, 1 mg/mL mouse IgG, 5 mg/mL BSA, 20% glycerol, pH 6.8) containingnone (Control) or 1 mM HEDTA was added to the beads. After 30-minutesincubation, the beads were washed again, a substrate solution for AP wasadded and the signal was detected with a luminometer. The signal wasmeasured when detection buffer was first prepared and after storage at37° C. for 2 days. Table 1 shows the percent (%) signal for the varioussamples at day 0 and day 2.

TABLE 1 Reagent TSI (IU/L) Control 1 mM HEDTA Signal at Day 0 (KCPS) 0459 43 0.9 502 160 4.6 582 579 39.9 1474 3693 AP Counts 5651 6275 Signalat Day 2 (KCPS) 0 247 41 0.9 239 134 4.6 252 364 39.9 236 2439 AP Counts5167 6548 Signal Recovery at Day 2 (%) 0 54% 97% 0.9 47% 84% 4.6 43% 63%39.9 16% 66% AP Activity 91% 104% 

The reagent with 1 mM HEDTA showed nearly an order of magnitude lowernon-specific binding (signal for zero TSI sample) and two fold highersignal than the control reagent at day 0 for the sample with the highestTSI concentration. The reagent containing HEDTA retained 66% of itsactivity compared to only 16% for the control reagent. The AP enzymaticactivity varied slightly between 91% and 104% for the two reagents,which demonstrates that the enhanced activity retention was due toimproved stability of the TSH receptor in the presence of HEDTA inaccordance with the principles described herein and not to improvedstability of AP.

Example 3 Effect of Glycerol in TSH Receptor Buffer

A TSH receptor chimera conjugated to AP (prepared as described above)was dissolved in detection buffers (0.1 M PIPES, 5 mM magnesium acetate,0.5 mM zinc chloride, 1 mM HEDTA, 5 mg/mL BSA, 10 mg/mL casein, 2.5μg/mL Amphotericin B, 0.2 mg/mL gentamycin sulfate) containing variousconcentrations of glycerol. The detection buffers were evaluated whenfirst prepared and after storage at 37° C. for 7 days. Table 2 shows thepercent (%) of initial signal retained after 7 days for the variousreagents.

TABLE 2 Glycerol Amounts TSI 20% 30% 40% 50% (IU/L) Percent (%) ResponseRetained after 7 Days at 37° C. 0.00 97% 114%  106% 105% 0.18 73% 102% 103% 106% 0.37 70% 98% 104% 101% 0.77 66% 97% 109% 103% 1.59 67% 102% 100%  97% 3.36 68% 104%  109% 104% 6.84 59% 93% 102% 108% 13.73 57% 94%109% 104% 26.39 54% 85% 101% 103% 50.71 51% 77%  90% 100% Average 66%96% 103% 103%

The stability of the detection receptor, as indicated by the retainedsignal, improved from 66% to nearly 100% as the glycerol content of thereagent increased from 20% to 50%.

Example 4 Effect of HEDTA Concentration in TSH Receptor Buffer

A TSH receptor chimera conjugated to AP (prepared as described above)was dissolved in a buffer (0.1 M PIPES, 5 mM magnesium acetate, 0.5 mMzinc chloride, 5 mg/mL BSA, 10 mg/mL casein, 30% glycerol, 2.5 μg/mLAmphotericin B, 0.2 mg/mL gentamycin sulfate) containing variousconcentrations of HEDTA. Table 3 shows the signal (KCPS) obtained forthe various samples at various HEDTA concentrations.

TABLE 3 HEDTA (mM) TSI (IU/L) 0.1 0.5 0.75 1.0 1.25 2 0 17 21 19 17 1515 0.58 NA 925 929 953 872 957 9.26 288 1865 1939 2051 1883 1942 AP16674 17192 14457 14704 14767 13772

The signal for the highest TSI sample increased dramatically as theHEDTA concentration increased. The alkaline phosphatase (AP) activitydecreased slightly at higher HEDTA concentration, which demonstratesthat the increased signal at higher HEDTA concentration is due to higherretained activity of the receptor and not AP activity.

Example 5 Effect of TSH Receptor in a Chelating Buffer

A TSH receptor chimera conjugated to alkaline phosphatase (prepared asdescribed above) was dissolved in various buffers (MES, PIPES, Citrate)at various pH levels. All of the buffer formulations contained 10%glycerol as described above. FIG. 1 shows the signal for a sample having33 IU/L of TSI. The signal varied from 2000 KCPS to 4000 KCPS for thevarious buffers at various pH levels.

FIG. 2 shows the percent signal retained after storing the detectionbuffers with the receptor at 37° C. for one day. The MES and PIPESbuffers, which are not in accordance with the principles describedherein, retained 25% or less of initial signal regardless of the pH. TheCitrate buffer, in accordance with the principles described herein,retained between 65% and 80% of the initial signal.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings in accordance with the principlesdescribed herein that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims. Furthermore, the foregoing description, for purposes ofexplanation, used specific nomenclature to provide a thoroughunderstanding of the examples. It will be apparent to one skilled in theart that the specific details are not required in order to practice theexamples described herein. Thus, the foregoing descriptions of specificexamples in accordance with the principles described herein arepresented for purposes of illustration and description; they are notintended to be exhaustive or to limit the examples to the precise formsdisclosed. Many modifications and variations are possible in view of theabove teachings. The examples were chosen in order to explain theprinciples described herein and their practical applications and tothereby enable others skilled in the art to utilize the teachings.

What is claimed is:
 1. A diagnostic assay reagent compositioncomprising: an aqueous medium; a first thyroid stimulating hormonereceptor bound to a label; a second thyroid stimulating hormone receptorbound to a support; a chelating agent in an amount of about 0.1 mM toabout 20 mM; and a C2-C6 polyol in an amount of about 5% to about 50% byweight; wherein the support is at least one particle, and wherein thelabel is selected from the group consisting of an enzyme, a dye, afluorescent molecule, a chemiluminescent molecule, a coenzyme, an enzymesubstrate, a radioactive group, a small organic molecule, and anamplifiable polynucleotide sequence.
 2. The diagnostic assay reagentcomposition according to claim 1, wherein at least one of the first andsecond thyroid stimulating hormone receptors is a thyroid-stimulatinghormone receptor chimera.
 3. The diagnostic assay reagent compositionaccording to claim 1, wherein at least one of the first and secondthyroid stimulating hormone receptors present in the composition has ahalf-life of at least about 12 months during storage at a temperature ina range of from about 2° C. to about 40° C.
 4. The diagnostic assayreagent composition according to claim 1, wherein the chelating agentcomprises a triacetic acid moiety or a salt thereof, a tetraacetic acidmoiety or a salt thereof, a pentaacetic acid moiety or a salt thereof,or a hexaacetic acid moiety or a salt thereof.
 5. The diagnostic assayreagent composition according to claim 1, wherein the chelating agent isselected from the group consisting ofN-(2-hydroxyethyl)-ethylenediamine-N,N′,N′-triacetic acid and its salts,ethylene diamine tetraacetic acid and its salts, and ethylene glycoltetraacetic acid and its salts.
 6. The diagnostic assay reagentcomposition according to claim 1, wherein the C2-C6 polyol is selectedfrom the group consisting of glycerol, propylene glycol, ethyleneglycol, erythritol, xylitol, ribitol, and sorbitol.
 7. A method ofpreparing a liquid solution of thyroid stimulating hormone receptors,the method comprising: combining in a liquid medium a first thyroidstimulating hormone receptor, a second thyroid stimulating hormonereceptor, a chelating agent, and a C2-C6 polyol, wherein the firstthyroid stimulating hormone receptor is bound to a label and the secondthyroid stimulating hormone receptor is bound to a support, and whereinthe chelating agent and the C2-C6 polyol is in an amount sufficient toachieve a stable and active receptor in the liquid solution; andmaintaining the liquid solution at a temperature of about 2° C. to about40° C.; wherein said amount of the chelating agent is of about 0.1 mM toabout 20 mM; and said amount of the C2-C6 polyol is of about 5% to about50% by weight; wherein the support is at least one particle, and whereinthe label is selected from the group consisting of an enzyme, a dye, afluorescent molecule, a chemiluminescent molecule, a coenzyme, an enzymesubstrate, a radioactive group, a small organic molecule, and anamplifiable polynucleotide sequence.
 8. The method according to claim 7,wherein at least one of the first and second thyroid stimulating hormonereceptors is a thyroid-stimulating hormone receptor chimera.
 9. Themethod according to claim 7, wherein the chelating agent comprises atriacetic acid moiety or a salt thereof, a tetraacetic acid moiety or asalt thereof, a pentaacetic acid moiety or a salt thereof, or ahexaacetic acid moiety or a salt thereof, and wherein the C2-C6 polyolis selected from the group consisting of glycerol, propylene glycol,ethylene glycol, erythritol, xylitol, ribitol, and sorbitol.
 10. Themethod according to claim 7, wherein the amount of chelating agent inthe liquid solution is about 0.1 mM to about 20 mM, and wherein theamount of the C2-C6 polyol is about 5% to about 50% by weight.
 11. Amethod of stabilizing a liquid solution of thyroid stimulating hormonereceptors, the method comprising: combining a liquid solution comprisinga first thyroid stimulating hormone receptor and a second thyroidstimulating hormone receptor with a stabilizing amount of both (i) achelating agent selected from the group consisting of triacetic acidchelating agents and tetraacetic acid chelating agents and (ii) a C3-C5polyol, and wherein the first thyroid stimulating hormone receptor has alabel bound thereto and the second thyroid stimulating hormone receptorhas a support bound thereto; wherein said stabilizing amount of thechelating agent is of about 0.1 mM to about 20 mM; and said stabilizingamount of the C2-C6 polyol is of about 5% to about 50% by weight;wherein the support is at least one particle, and wherein the label isselected from the group consisting of an enzyme, a dye, a fluorescentmolecule, a chemiluminescent molecule, a coenzyme, an enzyme substrate,a radioactive group, a small organic molecule, and an amplifiablepolynucleotide sequence.
 12. The method according to claim 11, whereinat least one of the first and second thyroid stimulating hormonereceptors is a thyroid stimulating hormone receptor chimera.
 13. Themethod according to claim 11, wherein the chelating agent is selectedfrom the group consisting ofN-(2-hydroxyethyl)-ethylenediamine-N,N′,N′-triacetic acid and its salts,ethylene diamine tetraacetic acid and its salts, and ethylene glycoltetraacetic acid and its salts.
 14. The method according to claim 11,wherein the amount of chelating agent in the liquid solution is about0.1 mM to about 20 mM.
 15. The method according to claim 11, wherein theamount of C3-C5 polyol is about 5% to about 50% by weight.
 16. A methodof detecting a thyroid stimulating hormone receptor antibody in asample, said method comprising: providing in combination in an assaymedium a sample suspected of containing a thyroid stimulating hormonereceptor antibody and the composition according to claim 1; examiningthe combination for the formation of a complex comprising the thyroidstimulating hormone receptor antibody, the first thyroid stimulatinghormone receptor bound to the label, and the second thyroid stimulatinghormone receptor bound to the support; and relating the presence of thecomplex to one or both of a presence and an amount of the thyroidstimulating hormone receptor antibody in the sample.